1. Field of the Invention
This invention relates to the fields of polymer chemistry, immunology and transplantation, and more particularly to the field of materials for use in conjunction with transplantation and implantation of foreign cells and biological materials.
2. Art Background
Evidence exists that transplantation of insulin-producing cells (islets) can cure the diabetic animal of the need for insulin therapy. The major obstacle preventing clinical success in islet transplantation as a therapy for diabetes to date has been immunogenicity of the cell and rejection of the transplanted graft. Survival of islet allografts and or xenografts has been achieved by various methods of immunosuppression and/or related immunological techniques. However, such techniques have had only limited success in that the transplanted islet cells survive only a short while before rejection occurs. In addition, the extended use of immunosuppressive agents often results in severe complications, such as, renal damage and even cancer in the transplant recipient.
One solution to this problem of graft rejection is the introduction of a physical, semi-permeable barrier between the transplanted islets and the host's immune system by the method of microencapsulation. Microencapsulation is a process in which small, discrete materials, viable biological tissue or cells, liquid droplets, or gases are completely enveloped by an intact membrane which is preferably compatible with the biological system in which it is placed. The function of the microcapsule membrane is to protect the material within from immunological recognition by the host and to control the flow of materials inside and outside the microcapsule across the membrane.
A large body of literature on microencapsulation has been produced including Darquy, S. and Reach, G. Diabetologia, (1985) 28:776-780; Lim, F. and Sun, A. Science, (1980) 210:908-910; Lim, F. and Moss, R. Journal of Pharmaceutical Sciences, (April, 1981) 351-354; O'Shea, et al. Biochemica et Biophysica Acta. 804 (1984) 133-136; Leung, et al. Artificial Organs, (1983) 7(2):208-212; Araki, et al. Diabetes, Vol. 34, September 1985, 850-854; and U.S. Pat. Nos. 4,690,682; 4,409,331; 4,391,909, among others.
In addition to islet cells, other materials such as tissue, charcoal, microbial cells, yeasts, chloroplasts, plant protoplasts, mitochondria and enzymes have been immobilized and entrapped using microencapsulation techniques.
Attempts have been made to transplant such encapsulated material into a patient to perform the specific function of that material inside the recipient patient. For example, activated charcoal could be used to detoxify blood, while pancreatic tissue could supplement the patient's supply of insulin. See, e.g., Lim and Sun (1980) Science 210, 908; O'Shea, et al. (1984) Biochim. Biophys. Acta 804, 133.
While such attempts have been partially successful, the patient's body often reacts in ways that impair the activity of the microcapsules by fibroblast overgrowth of this substance by the body. A potential mechanism for the induction of fibroblasts is the activation of macrophages, and the resultant stimulation of cytokines by the capsule substance. Cytokines are molecules secreted by the body in response to a new set of antigens, and are often toxic to the encapsulated cells. Some cytokines in turn stimulate the immune system of the patient. Thus, immune response can still be a limiting factor in the effective life of the encapsulated material.
In addition, fibroblast cells tend to overgrow the microcapsules, also apparently in response to the newly released cytokines. Dinerallo, in LYMPHOKINES AND THE IMMUNE RESPONSE (Cohen, ed. 1990) CRC Press, p. 156; Piela and Korn, in LYMPHOKINES AND THE IMMUNE RESPONSE (Cohen, ed. 1990) CRC Press, pp. 255-273. This growth of fibroblasts causes the microcapsules to lose their porosity. As a result, the cellular material inside the microcapsules cannot receive nutrients and the product of the cellular material cannot permeate the microcapsule wall. This can cause the encapsulated living material to die, and can impair the effectiveness of the microcapsules as a delivery system.
Among the materials used for encapsulation are calcium alginate gels. Lim and Sun, in 1980, successfully microencapsulated islets using alginate gel, poly-L-lysine and polyethylenimine. The encapsulated islets were injected intraperitoneally into diabetic rats. The animals' blood glucose levels dropped to normal for two to three weeks, suggesting that the encapsulation process had protected the islets from invasion by the recipients' immune system. However, these studies showed that the microcapsules were eventually rejected as a result of fibrosis, or fibroblast formation around the microcapsule, which eventually restricts the flow of nutrients to the cells contained in the microcapsule and the outflow of material from the microcapsules created by the islet cells disposed therein.
Alginate, the principal material of the microcapsules, is a heterogeneous group of linear binary copolymers of 1-4 linked .beta.-D-mannuronic acid (M) and its C-5 epimer .alpha.-L-guluronic acid (G). The monomers are arranged in a blockwise pattern along the polymeric chain where homopolymeric regions (M blocks and G blocks) are interspaced with sequences containing both monomers (MG blocks). The proportion and sequential arrangement of the uronic acids in alginate depend upon the species of algae and the kind of algal tissue from which the material is prepared. Various properties of different types of alginates are based upon the guluronic acid makeup of the particular alginate. For example, viscosity depends mainly upon the molecular size, whereas the affinity for divalent ions essential for the gel-forming properties are related to the guluronic acid content. Specifically, two consecutive di-axially linked G residues provide binding sites for calcium ions and long sequences of such sites form cross-links with similar sequences in other alginate molecules, giving rise to gel networks.
Commercial alginates are produced mainly from Laminaria hyperborea, Macrocystis pyrifera, Laminaria digitata, Ascophyllum nodosum, Laminaria japonica, Eclonia maxima, Lesonia negrescens and Saragassum sp.
Additionally, alginates may be obtained from certain bacteria. Azotobacter vinelandii produces O-acetylated alginate with a content of L-guluronic acid ranging from 15 to 90%. Pseudomonas aeruginose under certain growth conditions produces poly-mannuronic acid and such bacteria as well as certain other alginate producing Pseudomonades are not able to produce polymers containing G-blocks.
Also, alginates having high or low contents of G or M residues may be obtained from specific portions of algal tissue. For example, alginate having a high content of guluronic acid may be obtained from the outer cortex of old stipes of L. hyperborea. Alginate having a high content of guluronic acid can also be prepared by chemical fractionation or by enzymatic modification using mannuronan C-5 epimerase. This enzyme is able to introduce G-blocks into an existing alginate polymer, producing polymers with high G-block content.
It is believed that alginate itself is one of the materials of the microcapsules which causes fibrosis, such that attempted implantation or transplantation of alginate encapsulated material is viable only for a short term.
A measure of the potential to cause fibrosis can be obtained from the ability of certain substances to induce cytokine production, including tumor necrosis factor-.alpha. (TNF-.alpha.), interleukin-1 (IL-1) and interleukin-6 (IL-6). These cytokines play an important role in immune responses and in inflammatory reactions. These macrophage-derived mediators are known to regulate fibroblast proliferation (Libby et al., J. Clin. Invest. (1986) 78:1432; Vilcek et al., J. Exp. Med. (1986) 163:632). A possible mechanism for the fibrotic reaction to implanted microcapsules is the activation of macrophages, either by a contaminant within commercial alginate (e.g., polyphenols or lipopolysaccharides (LPS)), or by alginate monomers directly, with subsequent release of cytokines responsible for fibroblast migration and proliferation. LPS are known to stimulate the immune response. Additionally, polysaccharides other than LPS have been reported to have an immunostimulating effect, including antitumor activity and stimulation of monocyte functions. However, little is known about the effects of polysaccharides on cytokine production from monocytes.